The present invention is in the technical field of two-dimensional separation of proteins and other biologicals and relates particularly to apparatus and a method for the rapid and reproducible separation of species in a liquid medium.
The separation and characterization of proteins is ubiquitous throughout the life sciences. Two of the most popular electrophoresis separation techniques are: 1) gel isoelectric focusing (IEF), where the separation mechanism is based on protein surface charge providing isoelectric point (pI) separation and 2) sodium dodecyl sulfate (SDS) gel electrophoresis where the separation mechanism is based on molecular weight (MW). These two techniques are most commonly performed individually.
Isoelectric focusing (IEF) is a special electrophoretic technique for separating amphoteric substances such as peptides and proteins in an electric field, across which there is both voltage and a pH gradient, acidic in the region of the anode and alkaline near the cathode. Each substance in the mixture will migrate to a position in the separation column where the surrounding pH corresponds to its isoelectric point. There, in zwitterion form with no net charge, molecules of that substance cease to move in the electric field. Different amphorteric substances are thereby focused into narrow stationary bands.
In IEF separation, it is well known that proteins having molecular weight differences or conformational differences may possess similar pI values and therefore focus at the same location. In order to then separate these co-focused proteins, a technique called two-dimensional (2D) gel electrophoresis has been employed. 2D gel electrophoresis combines two orthogonal separation techniques—gel IEF and SDS gel—to create a technique that dramatically increases separation resolution and provides for the separation of co-focused IEF protein zones. 2D gel electrophoresis is generally carried out in a polyacrylamide slab gel and although it has become a workhorse in the field of proteomics, owing to the high degree of resolution which can be obtained thereby, it is very labour-intensive, time consuming and non-quantitative.
Moreover, although 2D gel electrophoresis does afford the highest degree of molecular weight resolution of known electrophoretic separation techniques, it has not yet been possible to automate that process nor quantify the resolved component proteins or other analytes. These and other drawbacks have motivated researchers to combine two orthogonal separation techniques in the liquid phase, using a capillary or coplanar microchannel format. While these are necessarily “limited resolution” techniques, relative to 2D gel electrophoresis, they are much simpler and faster to use and are of adequate resolution for many purposes.
It is known to combine capillary or channel isoelectric focusing (cIEF) with non-porous reverse phase microliquid chromatography (RPLC) in a two-dimensional layout, to obtain useful online detection and quantitation. However, the interface between the first and second separation dimension has hitherto been carried out only at the outlet end of the IEF separation capillary or channel. It is known that the separation and pH gradient obtained in cIEF may be disturbed when mobilizing focused protein zones to reach the outlet end. A as result, it is more challenging to transfer separated zones from the first separation dimension to the second separation dimension in the orthogonal capillary or microchannel format than in apparatus for 2D gel electrophoresis. Fluid connections and for control of nanoliter volumes are required, making for complex analytical design and operation.